The pyruvate dehydrogenase complex (PDC) functions at an important branch point for energy metabolism both in regard to utilization and storage of cellular fuels, carbohydrate and fat. Mammalian PDC is regulated by interconversion between an active and an inactive form by a specific kinase and phosphatase. Liver PDC catalyzes a rate limiting reaction in fatty acid synthesis and may respond to hormonal regulation. Brain PDC catalyzes a rate limiting reaction in the major energy pathway of the brain--glucose oxidation. We have purified liver PDC and isolated highly purified brain PDC. We will characterize the major determinants in the dynamic processes by which these interconvertible complexes are regulated. Many important features of the regulation of PDC can be detected only under conditions of the steady-state functioning of the complete monocyclic interconversion-cycle. Our investigation of this process should reveal which, among the multiplicity of effectors, have dominant roles in modulating this cycle and should characterize concerted effects of modulating ligands. Studies will test our hypotheses that the lipoyl moiety in PDC: a) has a mediatory role in the regulation of kinase activity by the NADH:NAD ion ratio, acetyl-CoA:CoA ratio and pyruvate concentration; and b) functions in inter-lipoyl transfer of acetyl groups and electrons between the subunits of the dihydrolipoyl transacetylase component which appear to contain two lipoyl moieties per subunit. Ca2 ion has been suggested as a second messenger for insulin induced activation of adipose and possibly liver PDC. An approach is proposed to determine whether the level of free Ca2 ion limits phosphatase activity under high energy conditions in rat liver mitochondria. We have demonstrated interconversion of PDC in the fat bodies of tobacco hornworms. We will characterize the effects of nutrition, development and in particular insulin-like polypeptides isolated from these insects on the level of active PDC.